Volume 1, Issue 5, Pages 729-739 (April 1998) Unveiling Two Distinct Ribonuclease Activities and a Topoisomerase Activity in a Site- Specific DNA Recombinase  Chong-jun Xu, Ian Grainge, Jehee Lee, Rasika M Harshey, Makkuni Jayaram  Molecular Cell  Volume 1, Issue 5, Pages 729-739 (April 1998) DOI: 10.1016/S1097-2765(00)80072-6

Figure 1 The General Mechanism for DNA Recombination by the Integrase Family Recombinases The reaction proceeds in two steps that are chemically identical. Each step involves the breakage of two DNA chains using active site tyrosines from recombinase monomers as the nucleophiles, followed by chain joining across partners using 5′-hydroxyl groups as the nucleophiles. The strand exchange region (thin gray lines) on each DNA partner is flanked by two Flp binding elements (parallel arrows) in inverted orientation. The phosphodiesters involved in recombination are indicated by (P). Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)

Figure 2 Strand Cleavage and Strand Joining by Flp in DNA Half-Sites and DNA-RNA Hybrid Half-Sites (A) Recombination within a DNA half-site (S) requires the concerted action of two Flp monomers, a shared active site being assembled at the dimer interface (Chen et al. 1992b; Lee and Jayaram 1995). Attack by Tyr-343 of one Flp monomer on the labile phosphodiester releases the trinucleotide (5′HOTTT3′). Phosphoryl transfer from the 3′-O-phosphotyrosine bond to the 5′-hydroxyl group of the bottom strand results in the formation of the hairpin recombinant R. The parallel lines including the C-G pair in bold letters represent the Flp binding element (5′GAAGTTCCTATAC3′/3′CTTCAAGGATATG5′). (B) Reactions were carried out using half-sites in which the top strand (the one that is cleaved by Flp) is labeled at the 5′ end (asterisk). A ribonucleotide position within the labeled strand is indicated by the 2′-OH, or by the T-to-U substitution. The molecular markers (M) are deoxyoligonucleotides containing 5′-phosphate and 3′-hydroxyl ends. They range in size from 32 to 14 nt, with a difference of 2 nt between two successive rungs of the ladder. (C) reactions were done with the indicated substrates and Flp(Y343F) in the absence or presence of tyramine (100 mM). In (B) and (C), the substrate and recombinant bands are indicated by (S) and (R), respectively. The cleavage products from the Flp or/and Flp(Y343F) reactions are denoted by (CP1), (CP2), and (CP3). In (A), (B), and (C), the phosphodiester position that normally participates in the recombination reaction is shown as (p). Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)

Figure 3 Characterization of the Cleavage Products from Half-Sites Labeled with 32P on the Top Strand at the 5′ Ends (A) Complete extension of the radioactive strand in the 5′ end–labeled substrate ([S]; the asterisk indicates the label) by Klenow polymerase would give rise to the product EP. If the strand were to be cleaved to expose a 3′-hydroxyl group, extension of the cleavage product would also yield EP. (B) The indicated substrate (containing a 2′-OH at the C position on the top strand) was reacted with Flp as described under Figure 2 to obtain 70%–80% of conversion into CP1. Production of R was inhibited almost completely by phosphorylating the 5′ end of the bottom strand using unlabeled ATP and polynucleotide kinase. DNA was recovered from the reactions, aliquoted into equal samples, and subjected to various treatments as indicated below the respective lanes. (PK) stands for polynucleotide kinase. (C) The reactions for obtaining CP2 were done with Flp and a half-site containing 2′-OH groups at the U positions on the top strand. The yield of CP2 was approximately 30%–40% of the input substrate. In (B) and (C), the bands corresponding to the substrate and the extension product are designated as (S) and (EP), respectively. The arrow indicates the dephosphorylation product derived from CP1 or CP2. Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)

Figure 4 Characterization of the Cleavage Products Obtained with Half-Sites Labeled near the 3′ End The two substrates were labeled (asterisk) by the Klenow polymerase reaction using [α-32P]-TTP in the absence of the other three dNTPs. After the cleavage reactions with Flp or Flp(Y343F), the recovered DNA was either directly fractionated by electrophoresis (lanes 3 and 5) or treated with polynucleotide kinase in the presence of unlabeled ATP (lanes 7 and 9) prior to fractionation. Molecular size markers (deoxyoligonucleotides) were run in lanes 1 and 10. Those in lane 1 contained 32P (asterisk) in the 3′-proximal phosphodiester bond, and a free 5′-hydroxyl group. They were obtained by alkaline hydrolysis of hybrid oligonucleotides containing ribonucleotide placements at desired positions. The markers in lane 10 contained 32P (asterisk) at the 5′ ends and hydroxyl groups at the 3′ ends. Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)

Figure 5 Topoisomerase Activity of Flp Reactions were done with plasmid substrates containing the normal 8-bp (pSP-8) or the artificial 20-bp (pSP-20) spacer between two Flp binding elements in a head-to-head orientation. After phenol-chloroform extraction and ethanol precipitation, the DNA was fractionated in 0.8% agarose gels and subsequently stained with ethidium bromide for viewing under UV light. Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)

Figure 6 Possible Mechanistic Paths for the Formation of CP1/CP1′ and CP2/CP2′ (A) Two possible mechanisms can, in principle, account for the formation of CP1/CP1′ from the substrate containing the ribo-C immediately 5′ to the scissile phosphodiester. In one scheme, the phosphodiester bond is cleaved by Tyr-343 of Flp or by the tyrosine mimic added in solution to form a 3′-O-phosphotyrosine bond. The 2′-hydroxyl group then attacks the phosphotyrosine bond to yield a 2′,3′-cyclic phosphate that is hydrolyzed to the 3′-phosphate product. In the other, Tyr-343 or the tyrosine mimic facilitates the direct attack of the 2′-hydroxyl group on the phosphodiester bond to yield the 2′,3′-cyclic product. (B) The CP2/CP2′ product pair is formed as a result of the attack of the 2′-hydroxyl group from U on the phosphodiester bond that is immediately downstream of the one involved in the reaction shown in (A). The resultant formation of 2′,3′-cyclic phosphate, followed by its hydrolysis, is akin to the pancreatic RNase mechanism. Molecular Cell 1998 1, 729-739DOI: (10.1016/S1097-2765(00)80072-6)